Microtitre plate hybridization system for detection of thermophilic Campylobacter rRNA

A microtitre plate nucleic acid probe hybridization system was developed for the detection of ribosomal RNA from thermophilic Campylobacter (Camp. jejuni, Camp. coli, Camp. lari and Camp. upsaliensis). A specific DNA probe obtained by amplification of 23S rRNA sequences using the polymerase chain reaction technique was immobilized on a microtitre plate, and used for hybridization with target 23S rRNA from cell lysates. The RNA-DNA hybrids thus formed in the wells were detected by an immunoenzymatic assay using a monoclonal antiRNA-DNA hybrid antibody. The sensitivity of this system was 2.7 x 10(4) cells ml(-1). This simple, sensitive and inexpensive hybridization and immunoenzymatic assay system should facilitate the detection of Campylobacter in food and clinical samples.     

Nanodiagnostic Method for Colorimetric Detection of Mycobacterium tuberculosis 16S rRNA

A nanodiagnostic method using nucleic acid sequence-based amplification (NASBA) and gold nanoparticle probes (AuNP probes) was developed for colorimetric detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of mycobacterial RNA by the isothermal NASBA process. The amplicons were hybridized with specific gold nanoparticle probes. The RNA–DNA hybrids were colorimetrically detected by the accumulation of gold nanoparticles. Using this method, 10 CFU ml^?1 of M. tuberculosis was detected within less than 1 h. Results obtained from the clinical specimens showed 94.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.     

The evaluation of viral load in patients with hemorrhagic fever with renal syndrome (HFRS) by real-time polymerase chain reaction

The system for real-time polymerase chain reaction was developed for detection of Puumala virus RNAand quantitative analysis of viral RNAload in clinical samples. Dynamics of viral load in blood samples from 12 patients with HFRS (from day 4 to day 17 of the disease) was studied. At day 4 viral RNA load varied from 4 x 10(4) to 1 x 10(6) copies/ml. During the period of observation viral load decreased by 10 - 200 times and was not detected in majority of patients to day 11 - 15 of the disease. The hantavirus persisted in blood of four patients to day 17 of the disease with viral load varied from 2.7 x 10(3) to 2 x 10(4) copies/ml. Developed method was characterized by highest sensitivity (100%) for HFRS detection from day 4 to day 8 of the disease.     

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Enrichment followed by quantitative PCR both for rapid detection and as a tool for quantitative risk assessment of food-borne thermotolerant campylobacters

As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10(2) to 10(7) CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 x 10(3) and 2 x 10(3) CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 10(1) to 1 x 10(7) (R(2) = 1.00) for the RotorGene and 10(3) to 10(7) (R(2) = 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (R(2)) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.     

Development of a method for concentration of rotavirus and its application to recovery of rotaviruses from estuarine waters.

As part of our studies on the ecology of human enteric viruses, an improved method for detection of rotaviruses in water was developed, and their presence in Galveston Bay was monitored. Samples (378 liters) of estuarine water adjusted to pH 3.5 and a final AlCl3 molarity of 0.001 were filtered through 25-cm pleated cartridge-type filters (Filterite Corp., Timonium, Md.) of 3.0- and 0.45-micron porosity. Adsorbed virus was eluted with 1 liter of 10% tryptose phosphate broth, pH 9.5. Primary eluates were reconcentrated to a final volume of 10 to 20 ml by a simple and rapid magnetic iron oxide adsorption and elution procedure. Two percent casein at pH 8.5 effectively eluted rotavirus from iron oxide. A total of 21 of 72 samples of water, suspended solids, fluffy sediments, and compact sediments collected in different seasons in Galveston Bay yielded rotaviruses. Recovery of rotaviruses varied from 119 to 1,000 PFU/378 liters of water, 1,200 PFU/1,000 g of compact sediment, 800 to 3,800 PFU/378 liters of fluffy sediment, and 1,800 to 4,980 PFU from suspended solids derived from 378 liters of water based on immunofluorescent foci counts on cover slip cultures of fetal monkey kidney cells.     

Development of a method for concentration of rotavirus and its application to recovery of rotaviruses from estuarine waters

As part of our studies on the ecology of human enteric viruses, an improved method for detection of rotaviruses in water was developed, and their presence in Galveston Bay was monitored. Samples (378 liters) of estuarine water adjusted to pH 3.5 and a final AlCl3 molarity of 0.001 were filtered through 25-cm pleated cartridge-type filters (Filterite Corp., Timonium, Md.) of 3.0- and 0.45-micron porosity. Adsorbed virus was eluted with 1 liter of 10% tryptose phosphate broth, pH 9.5. Primary eluates were reconcentrated to a final volume of 10 to 20 ml by a simple and rapid magnetic iron oxide adsorption and elution procedure. Two percent casein at pH 8.5 effectively eluted rotavirus from iron oxide. A total of 21 of 72 samples of water, suspended solids, fluffy sediments, and compact sediments collected in different seasons in Galveston Bay yielded rotaviruses. Recovery of rotaviruses varied from 119 to 1,000 PFU/378 liters of water, 1,200 PFU/1,000 g of compact sediment, 800 to 3,800 PFU/378 liters of fluffy sediment, and 1,800 to 4,980 PFU from suspended solids derived from 378 liters of water based on immunofluorescent foci counts on cover slip cultures of fetal monkey kidney cells.     

Improved bacteria detection by coupling magneto-immunocapture and amperometry at flow-channel microband electrodes

This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H(2)O(2)) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10(2)-10(8) cell ml(-1), with a limit of detection of 55 cells ml(-1) in PBS, without pre-enrichment steps. Furthermore, 100 cells ml(-1) could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.     

Rotavirus viremia and extraintestinal viral infection in the neonatal rat model

Rotaviruses infect mature, differentiated enterocytes of the small intestine and, by an unknown mechanism, escape the gastrointestinal tract and cause viremia. The neonatal rat model of rotavirus infection was used to determine the kinetics of viremia, spread, and pathology of rotavirus in extraintestinal organs. Five-day-old rat pups were inoculated intragastrically with an animal (RRV) or human (HAL1166) rotavirus or phosphate-buffered saline. Blood was collected from a subset of rat pups, and following perfusion to remove residual blood, organs were removed and homogenized to analyze rotavirus-specific antigen by enzyme-linked immunosorbent assay and infectious rotavirus by fluorescent focus assay or fixed in formalin for histology and immunohistochemistry. Viremia was detected following rotavirus infection with RRV and HAL1166. The RRV 50% antigenemia dose was 1.8 x 10(3) PFU, and the 50% diarrhea dose was 7.7 x 10(5) PFU, indicating that infection and viremia occurred in the absence of diarrhea and that detecting rotavirus antigen in the blood was a more sensitive measure of infection than diarrhea. Rotavirus antigens and infectious virus were detected in multiple organs (stomach, intestines, liver, lungs, spleen, kidneys, pancreas, thymus, and bladder). Histopathological changes due to rotavirus infection included acute inflammation of the portal tract and bile duct, microsteatosis, necrosis, and inflammatory cell infiltrates in the parenchymas of the liver and lungs. Colocalization of structural and nonstructural proteins with histopathology in the liver and lungs indicated that the histological changes observed were due to rotavirus infection and replication. Replicating rotavirus was also detected in macrophages in the lungs and blood vessels, indicating a possible mechanism of rotavirus dissemination. Extraintestinal infectious rotavirus, but not diarrhea, was observed in the presence of passively or actively acquired rotavirus-specific antibody. These findings alter the previously accepted concept of rotavirus pathogenesis to include not only gastroenteritis but also viremia, and they indicate that rotavirus could cause a broad array of systemic diseases in a number of different organs.     

Semliki Forest virus multiplication in clones of Aedes albopictus cells.

A total of 115 clones of Aedes albopictus cells were examined for their response to infection with Semliki Forest virus. Virus yield and cytopathology showed a bimodal distribution. More than 68% of the clones gave low yields of virus (between 8 x 10(6) and 2 x 10(8) PFU/ml) with no discernable cytopathology, and 30% gave high yields of virus (between 1 x 10(9) and 8 x 10(9) PFU/ml) and showed moderate to severe cytopathology. To determine the level at which restriction in virus growth occurs in the low-virus-producing clones, we compared the nature and extent of several virus-directed events in selected low-virus-producing clones with the same events in high-virus-producing clones. Specifically, we compared virus-specified polypeptide synthesis, positive- and negative-strand RNA synthesis, adsorption, uncoating, and transfection with virion 42S RNA. These studies showed that whereas events before negative-strand RNA synthesis and all subsequent virus-specified events were markedly reduced in the low-virus-producing lines, compared with the high-virus-producing lines. Thus, the restriction in virus growth in the low-virus-producing lines occurs at the level of synthesis of negative-strand RNA. The consequence of this restriction in an early step in the virus multiplication cycle is discussed in terms of the survival of invertebrate cells after alphavirus infection.     

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.     

轮状病毒NASBA检测研究

轮状病毒是世界范围内流行性胃肠炎暴发的重要病因。以患者粪便为样抽提轮状病毒RNA,在轮状病毒VP7高保守基因区段上设计引物,运用核酸序列依赖的扩增(NASBA)法进行检测,变性琼脂糖凝胶电泳和Northern杂交验证。NASBA预期的特异性产物为392bp,并在仅以目标核酸为模板或在浓度高达1μg/μL的非特异性核酸存在的混合模板中,均有清晰的目标带产生,表现出了很高的特异性。其灵敏度和RT-PCR相同甚至更高,可检测到50pg的核酸,并且当反应时间为3h时检测灵敏度最高。NASBA法扩增效率高、灵敏度高、快速易操作,尤其适用在基层单位推广应用。     

Modified Laminar Flow Biological Safety Cabinet

Tests are reported on a modified laminar flow biological safety cabinet in which the return air plenum that conducts air from the work area to the high efficiency particulate air filters is under negative pressure. Freon gas released inside the cabinet could not be detected outside by a freon gas detection method capable of detecting 10(-6) cc/s. When T3 bacteriophage was aerosolized 5 cm outside the front opening in 11 tests, no phage could be detected inside the cabinet with the motor-filter unit in operation. An average of 2.8 x 10(5) plaque-forming units (PFU)/ft(3) (ca. 0.028 m(3)) were detected with the motor-filter unit not in operation, a penetration of 0.0%. Aerosolization 5 cm inside the cabinet yielded an average of 10 PFU/ft(3) outside the cabinet with the motor-filter unit in operation and an average of 4.1 x 10(5) PFU/ft(3) with the motor-filter unit not in operation, a penetration of 0.002%. These values are the same order of effectiveness as the positive-pressure laminar flow biological safety cabinets previously tested. The advantages of the negative-pressure return plenum design include: (i) assurance that if cracks or leaks develop in the plenum it will not lead to discharge of contaminated air into the laboratory; and (ii) the price is lower due to reduced manufacturing costs.     

Microbiological status of piggery effluent from 13 piggeries in the south east Queensland region of Australia

AIMS: To assist in the development of safe piggery effluent re-use guidelines by determining the level of selected pathogens and indicator organisms in the effluent ponds of 13 south-east Queensland piggeries. METHODS AND RESULTS: The numbers of thermotolerant coliforms, Campylobacter jejuni/coli, Erysipelothrix rhusiopathiae, Escherichia coli, Salmonella and rotavirus were determined in 29 samples derived from the 13 piggeries. The study demonstrated that the 13 final effluent ponds contained an average of 1.2 x 10(5) colony-forming units (CFU) 100 ml(-1) of thermotolerant coliforms and 1.03 x 10(5) CFU 100 ml(-1) of E. coli. The Campylobacter level varied from none detectable (two of 13 piggeries) to a maximum of 930 most probable number (MPN) 100 ml(-1) (two of 13 piggeries). Salmonella was detected in the final ponds of only four of the 13 piggeries and then only at a low level (highest level being 51 MPN 100 ml(-1)). No rotavirus and no Erysip. rhusiopathiae were detected. The average log10 reductions across the ponding systems to the final irrigation pond were 1.77 for thermotolerant coliforms, 1.71 for E. coli and 1.04 for Campylobacter. CONCLUSIONS: This study has provided a baseline knowledge on the levels of indicator organisms and selected pathogens in piggery effluent. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge gained in this study will assist in the development of guidelines to ensure the safe and sustainable re-use of piggery effluent.